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K B Roy

Publications and source records attributed to K B Roy.

At least 19 recordsLinked to original sources
PubMed2000

BanI restriction endonuclease binds in the major groove of DNA: an inhibition kinetic study using substrates with mismatch basepairs.

Structural information on BanI-DNA interaction was obtained from simple inhibition kinetic assays using altered substrates. Self-complementary 13-mer oligodeoxynucleotides with or without mismatch basepairs in the BanI recognition sequence (GGPyPuCC) were synthesized. UV melting curves and CD spectra indicated double-stranded B-DNA structure for all the oligomers. Among the seven oligomers with recognition sequences, GGTACC, GGTGCC, GGTATC, GGCACC, GGAGCC, GGTAAC, and GGATCC, only the first two were cleaved with BanI. Kinetics of BanI cleavage of the native substrate was inhibited competitively by all of the other oligomers except the one with sequence GGCACC. From inhibition kinetic analysis in presence of a fixed concentration of the inhibitor, apparent K(m) and K(I) were determined. The data were analyzed in the context of alterations made in the hydrogen bonding potential in the major and minor groove of DNA within the recognition sequence due to basepair mismatches. Such analyses led to the conclusion that BanI, like BamHI, binds in the major groove and the central thymines make important contact with the protein.

Base Pair Mismatch↗
PubMed1999

Laser light scattering immunoassay for malaria.

Laser light scattering immunoassay (LIA) was proposed as a prospective diagnostic method for the detection of antibody (or antigen) by monitoring the agglutination of antigen (or antibody) coated carrier particles using dynamic light scattering (DLS) as probe. LIA is a very sensitive assay as it can detect microscopic immune complexes even when antibody (or antigen) level is low. A sizeable number of human sera collected from malaria endemic areas and hospitals have been analysed by ELISA using Pf parasite lysate or a RESA derived synthetic peptide as antigen parallel to LIA using Pf antigen coated polystyrene latex beads. Comparative analysis of data suggests LIA to be as good as ELISA and possibly better in terms of sensitivity and simplicity. LIA can be a simple and inexpensive immunoassay suitable for field use and mass application.

Antigens, Protozoan↗
PubMed1998

Excess nucleoside triphosphates (or zinc) allow recovery of alkaline phosphatase activity following refolding under reducing conditions.

The contribution of ATP and other nucleotides to the stabilization of non-native structures has been described for some proteins. We report here the effect of GTP, ATP, and their nonhydrolyzable analogues on the denaturation and renaturation of the enzyme Escherichia coli alkaline phosphatase. We show that GTP, ATP, and their nonhydrolyzable analogues considerably stimulate renaturation of AP in the presence of 2-mercaptoethanol where spontaneous renaturation is completely arrested due to reduction of S-S bonds. GTP is the most efficient inducer of reconstitution of the active site and appears to play a specific role besides being a substrate. The reconstituted protein was found to be in the reduced form despite having near-normal activity. The self-refolded oxidized form and the GTP-refolded reduced form had the same KM/kcat values and showed similar structural properties. We conclude that GTP can not only induce reconstitution of dimerization-competent monomers because of its substrate nature but also act as a modulator of the activity of AP. We also report here on the Zn2+-assisted reconstitution of E. coli AP under reducing condition. The prior formation of a disulfide bond for positioning the active site residues in the proper geometry is unnecessary under this condition.

Adenosine Triphosphate↗
PubMed1998

Laser light scattering immunoassay: an improved data analysis by CONTIN method.

Laser light scattering immunoassay (LIA) is a diagnostic method for the detection of antibody by monitoring the agglutination of antigen carrier particles mediated by antibody, using dynamic light scattering (DLS) as probe. We have used this method for the detection of antibody to P. falciparum that cause malaria. The data were analysed using CONTIN method and the superiority of the distribution analysis over the conventional interpretation of the data in terms of mean diffusion coefficient or hydrodynamic radius is discussed in detail.

Agglutination↗
PubMed1998

Protein engineering of BamHI restriction endonuclease: replacement of Cys54 by Ala enhances catalytic activity.

Chemical modification studies of BamHI endonuclease indicated the importance of the cysteine residue in catalysis [Nath, K. (1981) Arch. Biochem. Biophys, 212, 611-617]. Of the three cysteine residues at positions 34, 54 and 64 in the BamHI endonuclease Cys54 and Cys64 are at the DNA-protein interface. The co-crystal structure of the BamHI-DNA complex, however, does not indicate any role of cysteines either in binding or catalysis. In the context of strong biochemical evidence, Cys54 in BamHI was changed to Ala54 to investigate its role in catalysis. The mutation was carried out by PCR overlap extension, the mutant gene was cloned and characterized by sequencing. The mutant BamHI was expressed and purified to homogeneity and the kinetic parameters (K(M) and kcat) of the wild type and the C54A mutant were determined. The mutation results in up to approximately 40% enhancement of kcat and some increase in K(M). These in vitro results were also supported by in vivo SOS induction assays: the C54A mutant gene under the T7 promoter caused complete lysis in JH139 in absence of T7 RNA polymerase whereas the wild-type gene gave deep blue colonies under the same conditions. The results suggest no direct role of Cys54 in catalysis, but it can influence the catalytic activity through Val57 backbone contact seen in the co-crystal structure.

Alanine↗
PubMed1996

DNA recognition and structural specificities.

How a short DNA sequence interacts in a sequence specific manner with appropriate protein is understood only in certain systems for which high resolution crystal structures of the protein-DNA complexes are available. The base sequence of DNA is sensed directly (read-out) by the protein through the major or minor groove, while DNA shape also is sensed through multiple interactions with the sugar phosphate backbone. Several repressors, activators and restriction endonucleases complexed with their cognate DNA oligomers are now known and reviewed here. If the binding site on DNA has two fold symmetry, the protein interacts as dimer and uses a variety of structural motifs for specific interaction. The level of specificity of interaction is enhanced by flexibility and/or distortion in either the DNA or protein tertiary structure.

Animals↗
PubMed1995

Bam HI cleaves the self complementary dodecamer d-CGCGGAGCCGCG, before the two G's and possibly binds in the DNA major groove.

Oligodeoxynucleotides with GT or GA mispairs within the Bam HI recognition sequence (GGATCC), have been prepared. Binding and cleavage of the native vis a vis the mismatch substrates by Bam HI are analysed. UV melting curves and CD spectra of the oligomers suggest a double stranded B-DNA conformation. The enzyme Bam HI binds with varying affinities to the oligomers except the one with the GT wobble base pair. Bam HI cleaves the cognate sequence, GGATCC, between the two Guanines but cleaves GGAGCC before the guanines. The unusual cleavage is due to a local distortion in the DNA structure. Kinetic analysis of the cleavage reactions using the 35S labeled hexadecamers, d-ATGGCGGATCCGCCAT and d-ATGGCGGAGCCGCCAT, as substrates gives Km values 11.08 nM and 1.16 nM with corresponding Kcat of 11.04 and 0.62 min-1 respectively. The results are consistent with the binding of Bam HI in the major groove.

Base Composition↗
PubMed1994

Use of isotope-dilution phenomenon to advantage in the determination of kinetic constants Km and Kcat for BamHI restriction endonuclease: an empirical and iterative approach.

An assay using a very small amount of 35S-labeled deoxyoligonucleotide as a substrate for the determination of Km and Kcat for the restriction enzyme BamHI is described. Two synthetic deoxyoligonucleotides, ATGGCGGATCCGC and ATGGCGGAGCCGC, containing the cognate and a mismatch BamHI sequence, respectively, were labeled by an end-filling reaction using the Klenow fragment of DNA polymerase and [35S]dATP to generate the labeled self-complementary substrates. The dependence of BamHI hydrolysis on substrate concentration was investigated using mixtures of a fixed amount of radiolabeled substrate and varying amounts of cold-labeled substrate over a wide range. The apparent competitive inhibition observed due to the phenomenon of carrier dilution was analytically corrected by an empirical as well as an iterative approach to give Km values comparable to those reported in the literature. We have found that the values obtained using the empirical formula are very close to the precise values obtained through iteration. Our procedure has used isotopic dilution to advantage to make the assay less expensive and can be applied effectively to any enzyme-substrate reaction in which the substrate and the product have radioactive labels. The method would be especially useful for a rapid analysis and comparison of kinetic constants of various mutant enzymes or substrates.

Base Sequence↗
PubMed1994

Hairpin formation in d-AAGCTTAAGCTT under high salt conditions shows unusual properties.

The hairpin-duplex equilibria of the dodecamer d-AAGCTTAAGCTT and interaction of the duplex form with a pentapeptide, KGWGK, has been studied. UV thermal transitions are monophasic at low salt but biphasic at higher salt concentrations. At 10(-5) M or less oligomer concentration biphasic melting curves persist till 900 mM NaCl. The d(Tm)/d log(Na+) for the duplex form is 12 degrees C and for the hairpin is 18 degrees C. The delta H and delta S values for duplex formation are low (-25 K cal/mole and -59 Cal/mole respectively). KGWGK binds to the duplex form with a binding constant K = 3.4 x 10(5)M-1 measured from fluorescence quenching of tryptophan. These unusual results are markedly different from that reported for d-AGATCTAGATCT (Biochemistry 31, 6241-6245) and are discussed in terms of sequence dependence of loop folding and cruciform extrusion pathway of hairpin formation.

Amino Acid Sequence↗
PubMed1992

Binding of oligopeptides to d-AGATCTAGATCT and d-AAGCTTAAGCTT: can tryptophan intercalate in DNA hairpins?

The interactions of three tryptophan-containing peptides, KWK, KGWK tert-butyl ester, and KGWGK, with two self-complementary dodecamers of the same base composition but different sequence were studied by UV, CD, and fluorescence spectroscopy. The oligonucleotides, d-AGATCTAGATCT and d-AAGCTTAAGCTT, contain tandem repeats of the recognition site for the restriction enzyme BglII in the former and HindIII in the latter. Thermal transition data in dilute solutions and in 0.01 M NaCl indicate these dodecamers to be present in hairpin forms. Binding of peptides to these hairpins was followed by tryptophan fluorescence quenching titrations at 10 mM Na+; the data suggest intercalation of the indole ring. The association constants for the peptide-oligonucleotide (PN) complexes are an order of magnitude higher (10(5) M) than those reported with polynucleotides [10(4) M; Rajeswari et al. (1987) Biochemistry 26, 6825]. The pentapeptide, KGWGK, discriminates between BglII and HindIII sequences with higher affinity for the HindIII dodecamer. The CD maximum of KGWGK, at 220 nm, is drastically diminished upon interaction with oligonucleotides. The ellipticity at 220 nm is halved at 10 times less P/N ratio with the HindIII dodecamer than the BglII dodecamer, suggesting stronger binding to the HindIII dodecamer. The results are discussed in terms of two different modes of binding of oligopeptides to the DNA hairpins.

Amino Acid Sequence↗
PubMed1992

Hairpin and duplex forms of a self-complementary dodecamer, d-AGATCTAGATCT, and interaction of the duplex form with the peptide KGWGK: can a pentapeptide destabilize DNA?

Ordered forms of a synthetic dodecamer, d-AGATCTAGATCT, a direct repeat of the BglII recognition sequence, have been investigated using UV, CD, and fluorescence spectroscopy. Complex hairpin-duplex equilibria are manifest in UV thermal transitions, which are monophasic in the presence of very low or high NaCl concentrations but distinctly biphasic at intermediate ionic strengths. In 100 mM NaCl, the 1/Tm vs 1n C curve has a reasonable positive slope, which yields delta H and delta S for duplex formation as -66.2 kcal/mol and -190 cal/mol, respectively. Interaction of the dodecamer in duplex form with a tryptophan-containing peptide, KGWGK, has also been investigated to test the "bookmark" hypothesis (Gabbay et al., 1976) under the uniform structural constraint of the oligonucleotide of defined sequence. CD spectra of the peptide (P), the oligonucleotide (N), and their mixtures at different P/N ratios show a dramatic change in peptide spectrum but little change in nucleic acid dichroism with peptide binding. The Tm of P-N complexes decreases with an increase in peptide binding and levels off at saturation binding of P/N = 2.0. The data are interpreted in terms of a groove-cum-intercalation mode of binding, where intercalation to the tryptophan side chain destabilizes the double helix. A Scatchard plot of the binding data is nonlinear, with best-fit values for an overall association constant K = 4.33 x 10(5) M-1, and the number of binding sites n = 3.23 when fitted to the site-exclusion model of binding.

Amino Acid Sequence↗
PubMed1991

Fast and simple anion-exchange chromatography for large-scale purification of self-complementary oligonucleotides.

A fast and simple anion-exchange chromatography method is described for large-scale purification of synthetic oligonucleotides. Using a single matrix and aqueous solvent system, the two-step chromatographic procedure can handle complex separation problems of self-complementary or G-rich sequences without the use of urea or formaldehyde. The work also demonstrates the complication encountered, possibly due to hairpin formation, in one of the oligomers.

Bacterial Proteins↗
PubMed1986

Molecular conformation of gonadoliberin using two-dimensional NMR spectroscopy.

Complete resonance assignments of the proton NMR spectrum of gonadoliberin (in its native amide and free acid forms) have been obtained using two-dimensional nuclear magnetic resonance spectroscopy under three different environmental conditions, namely, dimethyl sulphoxide solution, aqueous solution and lipid-bound form in model membranes. The proton chemical shifts in the three cases have been compared to derive information about inherent conformational characteristics of the molecule. It has been inferred that the molecule possesses no short-range or long-range order under any of the three solvent conditions. However, there is a nonspecific increase in the linewidths when gonadoliberin is bound to model membranes, indicating a reduced internal motion in the molecule due to lipid-peptide interactions.

Deuterium↗
PubMed1985

Resonance assignment of the 500-MHz proton NMR spectrum of self-complementary dodecanucleotide d-GGATCCGGATCC: altered conformations at BamHI cleavage sites.

Resonance assignments of nonexchangeable base and sugar protons of the self-complementary dodecanucleotide d-GGATCCGGATCC have been obtained by two-dimensional NMR methods and strategies derived from interproton distance calculations on different secondary structures of nucleic acids. Conformational details about the glycosidic dihedral angle and sugar pucker have been derived from the relative intensities of cross peaks in the two-dimensional J-correlated and nuclear Overhauser enhancement correlated spectra in D2O solution. It is observed that d-GGATCCGGATCC assumes a predominantly B-type conformation with sequence-dependent changes along the chain. The recognition site of BamHI shows a distinctly different geometrical environment. The sugar rings of G1 and G7 assume a C3'-endo geometry while the rest of the sugars possess C2'-endo geometry.

Base Sequence↗
PubMed1983

A thermally driven interconversion of B and Z-dna.

CD and IR studies show that poly(dG-dMeC) in dilute Mg++ and Na+ solutions (e.g., 2 mM Mg++ and 55 mM Na+) undergoes reversible thermal interconversion of B and Z forms, in contrast to previous reports that the transition is independent of temperature and has an enthalpy change of zero. The B conformation is stable under the above conditions at lower temperature (approximately 5 degrees) and the Z form above approximately 20 degrees. The conversion is relatively slow in both directions, but is more rapid in the Z leads to B direction.

Circular Dichroism↗